Broad-spectrum histone deacetylase inhibitors (HDAC inhibitors or HDIs) are FDA approved drugs and important elements of modern targeted cancer chemotherapy. Adverse side effects are associated with these pan-HDIs, because they inhibit multiple HDACs with many different cellular functions. Selective inhibitors targeting specific HDACs are being developed to minimize the mechanism-based toxicity. Genetic analysis to define the functions of individual HDACs in basic cellular mechanisms is therefore essential to fully understand the mode-of-action of selective inhibitors prior to using them in cancer therapy. We showed HDIs kill cancer cells through a novel transcription-independent mechanism, which involves triggering DNA damage selectively in cycling cells. This provides a therapeutic window, and explains HDI action in selectively killing rapidly cycling cancer cells and not normal non-cancerous cells. Using novel selective inhibitors, we established a direct role for Hdac1 and Hdac2 (Hdacs1,2) in DNA replication and replication stress induced repair. Hdacs1,2, the key targets of FDA approved HDIs, control replication fork progression and genome stability in S-phase. Hdacs1,2 target histone acetylation to regulate nascent chromatin structure and remodeling during replication. Collectively, our studies provide a new paradigm for how we view HDAC and HDI actions in normal and cancer cells, and provide the basis for this proposal. In Aim 1, we will test the hypothesis that Hdacs1,2 via histone deacetylation control chromatin packaging and nucleosome structure to allow replication fork progression and maintain genome integrity. Roles for Hdacs1,2 in regulating the activities of a non-histone protein with important functions in DNA replication and repair via its deacetylation will also be explored. Understanding how HDACs contribute to efficient DNA replication and repair will provide a rational basis for the design and pre- clinical evaluation of selective HDIs that minimize unwanted toxicity while retaining clinical effectiveness. Acute lymphoblastic leukemia of early Pre-B-cell origin (Pre-B-ALL) is the most common pediatric cancer and accounts for 20% of acute leukemia in adults. Pre-B-ALL with the t(9;22) translocation (the Philadelphia (Ph) chromosome and coding for BCR-ABL) has the poorest clinical outcome and patients are at high risk of relapse with conventional therapies. BCR-ABL, the oncogenic fusion protein, enhances DNA repair to protect leukemic cells from apoptosis. In preliminary studies, selective inhibition of Hdacs1,2 caused death in Ph+Pre- B-ALL cells. Since Hdacs1,2 are required for replication and repair, we will test the hypothesis that selective inhibition of Hdacs1,2 overrides the BCR-ABL-driven efficient repair to compromise genome stability and cause death in Ph+Pre-B-ALL cells (Aim 2). Using mouse models, we will determine the functions for Hdacs1,2 in maintaining genome stability in early B-cell development and evaluate whether selective Hdacs1,2 inhibition is a viable therapeutic strategy for the treatment of early B-cell derived Pre-B-ALL. These studies will significantly advance cancer epigenetics and therapeutic fields, and provide a treatment option for a hard to treat cancer.